Medium and method of deriving and using a mutated bacteria strain

ABSTRACT

A new strain of  Bacillus anthracis  derived from the Sterne vaccine strain of  Bacillus anthracis  by growth on a high-nitrate-concentration, 3-amino-L-tyrosine growth medium.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.10/828,630, filed Apr. 9, 2004 (hereby incorporated by reference), whichclaims priority of the filing date of Provisional Application Ser. No.60/480,280, filed Jun. 20, 2003, the entire contents of which areincorporated by reference.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

BACKGROUND OF THE INVENTION

The present invention relates generally to the disease of anthrax and,more particularly, to a novel vaccine strain of Bacillus anthracis.Anthrax infections are initiated by spores of Bacillus anthracis, agram-positive, rod-shaped bacterium found in soil. Bacillus anthracisspores do not divide, have no measurable metabolism and are markedlyresistant to biological extremes of heat, cold, pH, desiccation,chemicals and irradiation. In the spore form, Bacillus anthracissurvives for decades, perhaps centuries. Domestic livestock are frequentvictims of the disease, but human cases of anthrax can occur as a resultof exposure to infected animals or animal products. Anthrax has alsobeen recognized as a likely biological warfare or terrorist agent.

Anthrax is a complex, poorly understood disease. The pathogenic processof anthrax and the mechanisms of immunity to the disease have not beencompletely defined. All known anthrax virulence genes are expressed bythe vegetative form of Bacillus anthracis that results from thegermination of spores within the body of the host. Spores introducedinto the body by abrasion, inhalation or ingestion are phagocytosed bymacrophages and carried to regional lymph nodes. Spores germinate insidethe macrophage and become vegetative bacteria. After germination andlocal multiplication within the macrophage, the vegetative bacteria killthe macrophage and are released into the bloodstream, reaching highnumbers (up to 10⁸ organisms per milliliter of blood) and causingmassive septicemia. It is believed that no immune response is initiatedagainst vegetative bacilli once they have been released from themacrophage.

It is believed that anthrax bacilli express a range of virulencefactors. The two major factors are a tripartite toxin and anantiphagocytic capsule composed of poly-D-glutamic acid. Anthrax toxinand capsule genes are apparently expressed early after germinationwithin the macrophage. The resulting toxemia and bacteremia havesystemic effects that lead to the death of the host.

The major virulence factors of Bacillus anthracis are encoded on twovirulence plasmids, pX01 and pX02. The toxin-bearing plasmid, pX01, is184.5 kilobase pairs (kbp) in size and codes for the genes (cya, lef,and pagA) that make up the secreted toxins. Regulation of toxinproduction is also encoded on pX01; it contains transacting regulatorgenes atxA and atxR.

The three proteins of the toxin are protective antigen (PA), lethalfactor (LF) and edema factor (EF). LF is a zinc metalloprotease thatinactivates mitogen-activated protein kinase kinase. EF is acalmodulin-dependent adenylate cyclase which causes fluid loss throughelevation of cellular cAMP concentrations in affected tissues. NeitherLF or EF are toxic alone; they can produce deleterious effects only whencombined with PA, so named because of its use in the protective anthraxvaccine. Following the A-B model of toxicity, PA serves as a necessarycarrier model for LF and EF and permits penetration into host cells.Lethal toxin, which results from the combination of LF+PA, stimulatesthe macrophages to release the shock-inducing mediators, necrosis factorα and interleukin-1β, which are partly responsible for sudden death insystemic anthrax. Edema toxin, which results from the combination ofEF+PA, is responsible for the massive edema seen in anthrax. Edema toxinalso plays a role in inhibiting phagocytic and oxidative burstactivities of polymorphonuclear leukocytes. Bacterial toxins thatincrease cAMP tend to decrease the immune response of phagocytes,thereby contributing to the development of infection.

The smaller capsule-bearing plasmid, pX02, is 95.3 kbp in size and codesfor the genes (capB, capC, capA) involved in the synthesis of thepolyglutamyl capsule. pX02 also encodes for a known transactingregulating gene for capsule modulation, acpA. atxA also appears toregulate acpA transcription to some degree.

The capsule is weakly antigenic and antiphagocytic. The toxins arethought to inhibit the immune response mounted against infection whilethe capsule inhibits phagocytosis of vegetative anthrax bacilli.

In addition to the major virulence factors already described, Bacillusanthracis likely expresses other plasmid—and chromosome—encoded genesthat contribute to the pathogenisis of the organism. Identification ofother genes contributing to virulence is crucial to the furtherdevelopment of effective protection against anthrax.

Expression of the known major virulence factors previously discussed(tripartite toxin and capsule) appears to be regulated by twohost-specific cues: elevated temperature and carbon dioxide/bicarbonateconcentration. During in vitro growth of Bacillus anthracis, synthesisof toxin protein and capsule is greatest when cultures are incubated atelevated (5% or greater) atmospheric CO₂ or when bicarbonate is added toculture medium in a closed vessel. Toxin and capsule synthesis is alsoincreased when cultures are incubated at 37° C. compared to when theyare incubated at 28° C. CO₂/bicarbonate and temperature—controlled geneexpression is at the level of transcription. As indicated previously,regulation of the expression of the toxin and capsule genes is mediatedby the transcriptional activator atxA; expression of the capsule gene isalso controlled by transcriptional regulator acpA.

The effect of these signals (CO₂/bicarbonate concentration andtemperature) in culture medium may be compared with their physiologicalrole in mammalian hosts; concentrations of CO₂ and bicarbonate in humansare similar to those that activate toxin and capsule production invitro, and the same is true of human body temperature. It is believedthat these signals play similar roles in vitro and in vivo by providingan optimal environment for expression of known Bacillus anthracis toxinand capsule genes.

As indicated previously, the loss of either plasmid pX01 or pX02 resultsin a marked reduction of virulence. This forms the basis for effectivevaccine production. Historically, vaccine strains of anthrax bacteriawere made by rendering virulent strains free of one or both plasmids.Pasteur, a heat-attenuated, pX02-carrying strain is encapsulated butdoes not express toxin components (pX01−/pX02+). Sterne, an attenuatedstrain that carries pX01, can synthesize toxin but does not have acapsule (pX01+/pX02−).

It is frequently convenient to class Bacillus anthracis with the“Bacillus cereus group” of bacilli which on the basis of phenotypecomprises Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis,and Bacillus mycoides. Except for Bacillus anthracis, all members ofthis group are resistant to penicillin. Bacillus anthracis is easy todifferentiate from other member of the Bacillus cereus group byobserving the morphological features of the colony on nutrient or bloodagar plates. Colonies of most Bacillus anthracis isolates have a mattappearance, are fairly flat, markedly tacky, white or grey-white andnon-hemolytic on blood agar and often having curly tailing at the edges.The unusually tenacious colonies are able to retain their shape whenmanipulated; disturbed sections of the colony often stand up like“beaten egg whites.” Bacillus anthracis is non-motile, sensitive topenicillin and the diagnostic Chemy gamma phage and able to produce thecapsule in blood or on nutrient agar containing 0.7% bicarbonatefollowing incubation in a 5-20% CO₂ atmosphere.

In practical terms, the demonstration of virulence constitutes theprinciple point of difference between typical strains of Bacillusanthracis and those of other members of the Bacillus cereus group.However, there is evidence that the virulence plasmids can betransferred between the Bacillus cereus group species through geneticengineering, although it is not clear how stable the resulting hybridsare.

An anthrax vaccine for humans is approved for use in the United Statesby the Food and Drug Administration. Designated anthrax vaccine adsorbed(AVA), it is an aluminum-hydroxide-precipitated preparation of PA fromattenuated, nonencapsulated Bacillus anthracis cultures of the Sternestrain. The anthrax vaccination protocol consists of 3 subcutaneousinjections given 2 weeks apart followed by3 additional subcutaneousinjections given at 6, 12 and 18 months. Annual booster injections ofthe vaccine are required to maintain immunity. Mild local reactionsconsisting of slight tenderness and redness at the injection site canoccur in approximately 30% of recipients. Severe local reactions occurinfrequently and consist of extensive swelling of the forearm inaddition to the local reaction. Systemic reactions characterized byflu-like symptoms occur in fewer than 0.2% of vaccines.

Animal studies have shown that AVA affords protection againstinhalational anthrax and a limited trial of a similar vaccine in humansindicated that it afforded considerable protection against cutaneousanthrax. Studies have also demonstrated, however, that the live Sternespore veterinary vaccine is more protective than the human chemicalvaccine. The enhanced protection conferred by the live vaccine probablyresults from stimulation of the host cellular immune system concurrentwith the humoral response to PA. The main limitation of the Sternevaccine is safety. Its use is sometimes associated with tissue necrosisat the site of inoculation and there have been rare fatalities. Becauseof these safety concerns, spore vaccines have generally not been usedfor humans.

The established virulence factors of Bacillus anthracis have been thetargets of most attempts to develop vaccines. As indicated previously,PA is asserted to be the essential anthrax-derived antigen for theprotective action of the current vaccine. Nevertheless, studies haverepeatedly demonstrated that titers to PA do not correlate strictly withthe level of immunity to anthrax. Moreover, it is important to note thatantibodies to PA induced by the vaccine are directed against the actionof the toxin and not at the multiplying Bacillus anthracis in aninfection. It has also been postulated that Bacillus anthracis strainscould be created by adding foreign genes from other toxic organisms. Asindicated previously, studies have shown that virulence plasmids can betransferred between the Bacillus cereus group of organisms.

Clearly there is a need for new candidate antigens for vaccinedevelopment, especially those that act prior to expression of anthraxtoxins into the body. Such vaccines should also be effective againstinfection with strains that have been engineered with additional toxins.

Critical to development of effective protection against anthrax is anunderstanding of the initial pathogenesis of the disease and itsvirulence mechanisms. Events occurring during the initial moments whenbacterial pathogens first encounter the host are critical for successfulestablishment of infectious loci. The pathogenesis of anthrax appears tobe related primarily to the unique sensitivity of the macrophage to theactivity of lethal toxin, in addition to the adenylate cyclase activityof edema toxin and the antiphagocytic properties of the capsule. Asindicated previously, the genes for these virulence factors are inducedin response to specific host-related cues, that is, CO₂/bicarbonatelevels and physiological body temperature. There is a need for a vaccinedirected to Bacillus anthracis targets vital for early steps in theinfection process, containing antigens which elicit antibodies targetedat the spore or germinating cell.

It is therefore a principal object of the present invention to provide avaccine strain of Bacillus anthracis from which may be produced animproved anthrax vaccine which is safe, nonreactogenic, efficaciousagainst genetically engineered strains, and which requires a minimalnumber of injections to achieve and maintain long-term immunity. It is afurther object of the invention to provide a vaccine strain of Bacillusanthracis that will enable identification of new genes that contributeto the pathogenesis of the organism and thereby elucidate new antigensthat play a role in eliciting a specific, protective immune responseearly in the infection process.

SUMMARY OF THE INVENTION

In accordance with the foregoing principles and objects of theinvention, a new strain of Bacillus anthracis is described which isderived from the Sterne vaccine strain of Bacillus anthracis by growthon a high-nitrate-concentration, 3-amino-L-tyrosine growth medium. Thenew strain, designated the Bacillus anthracis Alls/Gifford (Curlicue)strain, has a number of unique characteristics that are important indesigning a vaccine to restrict the growth of Bacillus anthracis inhuman or animal hosts.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be clearly understood from the following detaileddescription of preferred embodiments thereof read in conjunction withthe accompanying drawing wherein

FIG. 1 illustrates the sensitivity of Bacillus anthracis (Sterne strain)to heating.

FIG. 2 illustrates the sensitivity of the Alls/Gifford (Curlicue) strainto heating.

FIG. 3 illustrates the thermal response of Sterne strain at 125° C. onblood agar and 4×3×AT media in the absence of CO2.

FIG. 4 illustrates the thermal response of Sterne strain at 125° C. onblood agar and 4×3×AT media in the presence of CO2.

FIG. 5 illustrates time to death (TTD) of laboratory mice followinginfection with Sterne strain and Alls/Gifford (Curlicue) strain.

DETAILED DESCRIPTION OF THE INVENTION

In the inventors' efforts to ascertain the metabolic factors thatdetermine the progress of Bacillus anthracis through its life cycle, theinventors have examined various preparations of the special acceleratinggrowth medium 3-amino-L-tyrosine (3AT). This medium has been shown toaccelerate germination, growth, and sporulation of Bacillus anthracis inpreference over other bacilli species. 3AT is described in U.S. Pat.Nos. 5,156,971 and 5,003,050.

Nitration of Bacillus anthracis (and other microbes) is believed tocause DNA damage through guanine nitration, depurination and strandbreaking. This process can be at least partially repaired in cells thatare not killed out right; such repair can lead to mutation. The mutantBacillus anthracis Alls/Gifford (Curlicue) strain appeared in a modifiedhigh nitrate-concentration 3AT growth medium. This modified 3AT mediumwas composed of 55 g of trypticase soy broth (TSB) base, 12 g potassiumnitrate, 100 mg luminol (5-amino-2 3-dihydro-1,4-phthalazinedione), and80 mg 3-amino-L-tyrosine dihydrochloride per liter of water. The mediumwas inoculated with spores of the Sterne strain of B. anthracis derivedfrom the anthrax spore vaccine (Thraxol-2) manufactured by MobayCorporation. Fifty microliters of the spore suspension were placed in 5ml of TSB and pre-incubated for 2 hours to allow for germination. Fiftymicroliters of the germinated suspension were added to 100 ml of themodified 3AT broth medium and allowed to incubate overnight. After 24hours of growth, the broth cultures were plated onto sheep blood agarand 4×3AT agar plates (4× contains 2× the ingredients of 2×3AT agar).After 48 hours of incubation, small colonies were removed andtransferred to blood agar for an additional 24 hours of incubation.Spores were produced from the mutant (small colonies) by taking inoculumfrom solid media or liquid media and transferring it to blood agarplates and incubating at 37° C. for 4 days. The spores were harvestedfrom the agar plates with wet sterile cotton tipped swabs, which were,in turn, placed in sterile water. Using a sterile funnel, vacuum flask,and filter paper, vacuum collection was made of spores passed throughthe filter paper in the funnel. The filtrate was centrifuged and thebutton collected, which contained the pure spores. Bacillus anthracisAlls/Gifford strain is currently on deposit at the American Type CultureCollection under the designation PTA-3162.

Applicants have made available to the public without restriction adeposit of Bacillus anthracis ag (Alls/Gifford) with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110,U.S.A., ATCC Deposit No. PTA-3162. The date of the deposit was the 8thof March, 2001. The deposit with the ATCC was taken from the samedeposit maintained by the Air Force, since prior to the filing date ofthis application. All restrictions imposed by the depositor on theavailability to the public of the deposited material will be irrevocablyremoved upon the granting of the patent. The deposit of the Bacillusanthracis ag (Alls/Gifford) without restriction will be maintained atthe ATCC Depository, which is a public depository, for a period of 30years, or five years after the most recent request, or for the effectivelife of the patent, whichever is longer, and will be replaced if itbecomes nonviable during that period.

Bacillus anthracis Alls/Gifford has been propagated in culture and itscharacteristics, phenotypic and genotypic, examined. Upon microscopicexamination, the bacteria are long and filamentous initially like otherstrains of Bacillus anthracis, but curl and tightly coil into “knots”when the nutrients in the medium are depleted or the microbe is grown inmedium containing bicarbonate or carbon dioxide. Branching is seenunlike other strains of Bacillus anthracis. This strain is a slow growerthat produces pinpoint colonies on blood agar and 3AT medium. Thebacteria does produce viable spores but their production is delayed whencompared to other strains. As demonstrated in the following examples,Alls/Gifford, like Sterne, contains the pX01 plasmid, producesluminescent polymer diazoluminomelanin (DALM) when grown in 3AT medium,is penicillin sensitive, is lysed by Chemy gamma phage, isnon-hemolytic, and produces nitrite from nitrate. The examples alsoillustrate that Alls/Gifford does not show growth sensitivity to heatand bicarbonate (carbon dioxide) on 3AT medium, as does Sterne strain,and is less lethal than Sterne.

A genetic fingerprint comparison of the mutant Alls/Gifford with thepaternal Sterne strain should reveal the altered genes of the mutant.The protein products of the altered genes found by comparison to Sternecould form the basis for a vaccine that would stimulate antibody toinhibit the bicarbonate/CO2/heat-stimulated growth of anthrax that isnecessary for its development in the host. The genes will likely includechromosomal genes that cannot be easily altered by genetic engineeringto avoid vaccine protection, without compromising the survivability andpathogenicity of the anthrax. This vaccine could be produced in anexpression vector in E. coli or become a naked DNA vaccine that nolonger requires the whole anthrax or a supernatant derivative toproduce. The new vaccine should show decreased side effects and betterefficacy in generating an immune response (fewer inoculations). Becausegermination of spores and growth of the vegetative state should beeffected by the antibody induced by the vaccine, it should providebetter protection against larger doses of spores than the PA basedvaccines.

The following examples illustrate the invention:

Example 1 Genomic DNA Preparation and Polymerase Chain Reaction (PCR)

Bacillus anthracis Alls/Gifford (Curlicue) strain was subjected topolymerase chain reaction (PCR) to confirm the presence of the pXO1plasmid. First, chromosomal DNA was prepared from Bacillus anthracisAlls/Gifford as follows. A single colony of Bacillus anthracisAlls/Gifford was isolated from a tryptic soy (TSB) agar plate. Thecolony was used to inoculate 2 ml of TSB, was incubated overnight at 37°C. and next day was used to inoculate 100 ml of TSB, which was incubatedfor 2 days at 37° C.

The cells were harvested by centrifugation at 8,000 rpm in a Sorvall RC5B and SS34 rotor at 4° C. The pellet was resuspended in 10 ml 0.32Msucrose, 10 mM Tris HCl pH 7.5, 5 mM MgCl₂ solution, and left on ice for15 min. The suspension was centrifuged as described above. Aftercentrifugation the supernatant was poured off. Resuspension of thepellet was accomplished in 4.5 ml 0.075M NaCl, 0.024M EDTA solution, 0.5ml 5% SDS and 100 μl Proteinase K (10 mg/ml). The suspension was mixedand left overnight at 37° C. After incubation, 2.5 ml of phenolequilibrated with DNA buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA) wasadded and the mixture was shaken vigorously, centrifuged briefly, and2.5 ml chloroform/isoamyl alcohol (24:1 v/v) was added. The mixture wasshaken vigorously and centrifuged at 2,500 rpm for 5 min at roomtemperature. The upper aqueous layer was removed to a clean tube andreextracted with 5.0 ml chloroform/IAA. After shaking, the mixture wascentrifuged at 2,500 rpm for 2 min and the top layer remove to a cleantube. To precipitate the DNA 2.2 vol of ice-cold ethanol and 1/10th vol3M sodium acetate were added and the solution mixed by inversion. Theresulting spooled DNA was removed with a sterile tip and dissolved inDNA buffer. The concentration was calculated from reading 1 μl at260/280 nm with a Spectronic Genesys 5 spectrophotometer.

Bacillus anthracis Alls/Gifford DNA (Ba a/g) was diluted to 50 ηg/μl andsubjected to polymerase chain reaction (PCR) to confirm that the pXO1plasmid was still present, and that this was a mutant form of Bacillusanthracis. The PCR reaction mix contained 10×PCR buffer (PGCScientific), 2.6% DMSO, 2 mM dNTPs, 2 U Gene Choice TAQ polymerase (PGCScientific), 200 nM each BAPANTI Forward and BAPANTI Reverse2 primers(Genosys) and 5 μl of diluted Ba a/g DNA in a 50 μl reaction volume. Theprimers were designed to specifically detect the pa antigen gene (pag)carried on the pXO1 plasmid. PCR conditions using a Perkin Elmer 9600were 96° C. for 2 min, then 94° C. for 1 min, 60° C. for 1 min, 72° C.for 1 min for 35 cycles, followed by 72° C. for 5 min. The size of thePCR product was 959 bp. Using BAPANT Reverse1 primer a smaller productof 459 bp is formed

Fragment Forward sequence Reverse sequence  size atcaccagaggcaag R1tgtaattggagtaga 459 bp acacccccttgtggc actgaaatcgtcttg R2gctaactgattcttg 959 bp atattttgagatgttAn aliquot of 5 μl from the reaction mixture was subjected toelectrophoresis on a 0.8% agarose gel using TAE buffer. Bands werevisualized with Ethidium bromide.

Example 2 DALM Synthesis

The Alls/Gifford (Curlicue) strain, like the Sterne strain, synthesizesdiazoluminomelanin (DALM). DALM is a luminescent polymer. DALM can beused for chemiluminescent immunoassays for biological and chemicalagents; in radiofrequency and ionizing radiation dosimeters; and forRNA/DNA hybridization assays for viruses and genetic detection. DALM isdescribed in U.S. Pat. Nos. 6,013,520, 5,902,728, 5,156,971 and5,003,050.

To produce DALM, a modified 3AT medium composed of 55 g of trypticasesoy broth (TSB) base, 12 g potassium nitrate, 100 mg luminol (5-amino-23-dihydro-1,4-phthalazinedione), and 80 mg 3-amino-L-tyrosinedihydrochloride per liter of water was used. The medium was inoculatedwith spores of the Sterne strain of B. anthracis derived from theanthrax spore vaccine (Thraxol-2) manufactured by Mobay Corporation.Fifty microliters of the spore suspension were placed in 5 ml of TSB andpre-incubated for 2 hours to allow for germination. Fifty microliters ofthe vegetative suspension was added to 100 ml 4×3AT broth and wasincubated at 37° C. for 48 hr. The solution was transferred to 15 mlpolystyrene screw capped tubes and frozen at −20° C. overnight. Thetubes were removed from the freezer and the DALM, which was releasedfrom the cells and floated to the top, was removed from the top of thefrozen aqueous debris. The presence of DALM in the brown supernatant wasconfirmed by thermochemiluminescence in a Turner 20E luminometer in amixture of 100 μL of supernatant, 100 mircroliters of 0.3 M sodiumbicarbonate and 100 μL of 0.3% hydrogen peroxide heated to 45° C.Luminescent units produced were compared to a reagent blank containingno supernatant.

Example 3 Sensitivity to Penicillin

The Alls/Gifford (Curlicue) strain, like the Sterne strain, is sensitiveto penicillin. Using techniques well known in the art blood plates werespread with a suspension of vegetative Bacillus anthracis in TSB andpenicillin impregnated disks containing 10 U of penicillin were placedon the plate. The plate was incubated at 37° C. and observed. After 5 to6 hours a clear area was observed extending from the disk showingsensitivity of the Bacillus anthracis to the penicillin. Observing theBacillus anthracis cells at the edge of the cleared area microscopicallyshowed the cells to be aligned end to end and rounded in what isdescribed as “string of pearls” formation. This observation is one ofthe definitive tests for the presence of Bacillus anthracis.

Example 4 Cherry Gamma Phage

The Alls/Gifford (Curlicue) strain, like the Sterne strain, is lysed bythe Chemy gamma phage. Using techniques well known in the art blood agarplates were spread with a suspension of Bacillus anthracis vegetativecells in TSB and the plate incubated at 37° C. for 3 to 4 hour. Asuspension was made and 100, 50, 20, and 10 μL aliquots were dropped onto the lawn of Bacillus anthracis. The plates were incubated overnightand observed. Clear areas could be seen in the bacterial lawn where theChemy gamma phage suspension had been applied. By reducing the volumeapplied the titer of the phage suspension could be determined. This testis definitive for the presence of Bacillus anthracis.

Example 5 Non-Hemolytic

The Alls/Gifford (Curlicue) strain, like the Sterne strain, isnon-hemolytic. Using techniques well known in the art a suspension ofthe Alls/Gifford strain in TSB was spread on a blood agar plate andincubated at 37° C. overnight. The blood plate showed growth of thebacteria, but no haemolysis of the blood was observed

Example 6 Production of Nitrite from Nitrate

The Alls/Gifford (Curlicue) strain, like the Sterne strain, producesnitrite from nitrate. Cells were grown in 2.0 ml TSB overnight. Fiftymicroliters of this suspension were added to 2.0 ml of 4×3AT medium andallowed to incubate overnight at 37° C. The suspension was spun topellet the cells, the 50 μL of the supernatant removed to a clean tubeand 50 μL of Griess reagent A and 50 μL of Griess reagent B were added.A pink color was observed indicating the presence of nitrite.

Example 7 Thermal Sensitivity

The thermal sensitivities of Sterne and Alls/Gifford were compared.First, single-spore suspensions of Sterne were prepared as follows: TheSterne spore vaccine was centrifuged, the supernatant decanted, and thepellet washed with chilled deionized water. Dilute powdered milksolution was made with chilled deionized water to a concentration of 26mg of milk solids per ml of sterile milk solution. Fifty microliters ofthis suspension were diluted with 450 μL of physiological phosphatebuffered saline (PBS) and used as the source of colony forming unit(CFU) assays. Three microliters of the well suspended spore/skim milksuspension were transferred to the tip of a siliconized sterile pipet.The suspension was frozen and lyophized for four to five days. Thepipettes, charged with spores, were stored under vacuum at roomtemperature when not needed.

Single-spore suspensions of Alls/Gifford were prepared as follows: Fiftymicroliters of Bacillus anthracis Spore Vaccine, Thraxol-2, manufacturedby Mobay Corporation was grown in 5.0 ml of TSB and pre-incubated for 2hours (to allow for germination). Fifty microliters of the germinatedsuspension was added to 100 ml of 3AT medium containing 55 g TSB, 12 gPotassium nitrate, 100 mg Luminol, 80 mg 3AT per liter and allowed toincubate overnight. After 24 hour of growth the broth was plated toblood agar plates and 4×3AT solid medium. Small colonies were harvestedfrom the plates after 24 hr and replated on blood agar plates. Colonieswere allowed to grow on blood agar plates for 48 hours when they wereharvested into 4×3AT medium and spun. The pellet was washed in chilleddeionized water, spun and resuspended in powdered dry milk solution (26mg/ml). Fifty microliters of this suspension were diluted with 450 μL ofphysiological phosphate buffered saline (PBS) and used as the source ofcolony forming units (CFUs) for the assays. Three microliters of thewell-suspended spore/skim milk suspension were transferred to the tip ofa siliconized sterile pipet. The suspension was frozen and lyophized forfour to five days. The pipettes, charged with spores, were stored undervacuum at room temperature when not needed.

The lyophilized Sterne and Alls/Gifford spore samples were heated for 1second at various temperatures by placing them in the heating block ofthe melting point apparatus, which had been preheated to the desiredtemperatures. The pipettes were washed with 450 μL of sterile PBS. Thespores were plated on either blood or 4×3AT agar for 24 hours. A platereceived either 1 μL of the recovered spores by using a calibrated loopor 50 μL of the suspension. The plates were incubated overnight at 37°C. and counted the next day for colonies. In FIG. 1, the sensitivity ofSterne to heating is demonstrated. Starting at about 180° C. on bloodagar (indicated by circles), Sterne is affected by its exposure to heat.At 275° C., a 1 second exposure has killed all the Sterne spores. Sternegrown on 4×3AT media (as indicated by squares) is less viable than thatgrown on blood agar because the 4×3AT media is more stringent toward thegrowth of Bacillus anthracis. Sterne on 3AT is affected by heat startingat about 130° C. Sterne is no longer viable after a 1 second exposure at280° C.

The thermal resistance of Alls/Gifford is demonstrated in FIG. 2.Starting at about 40° C., on blood agar, Alls/Gifford is affected by itsexposure to heat (1 μL of inoculum on blood agar, as indicated bycircles). Fifty microliters of Alls/Gifford grown on 4×3AT media (asindicated by diamonds) is affected by heat starting at about 240° C.Results for 1 μL of inoculum on 4×3AT media (as indicated by triangles)and 50 μL of inoculum on blood agar (as indicated by squares) are alsoillustrated. At 300° C., a 1 second exposure has killed all of theAlls/Gifford spores on both media. Comparison of FIGS. 1 and 2 indicatethe increased sensitivity of Sterne to heat, and relative thermalresistance of Alls/Gifford. To further confirm the thermal resistance ofAlls/Gifford, using techniques well known in the art, attempts were madeto cure Alls/Gifford of its pX01 plasmid by 10 passages and cultivationover many days at 42° C. These attempts failed. By contrast, when Sternestrain is grown at an elevated temperature (42° C.) for ten days andpassaged to new medium every 24 hours, Sterne is cured of its pX01plasmid.

FIGS. 3 and 4 demonstrate the alteration of the thermal sensitivity ofSterne strain with CO2 or bicarbonate on 4×3AT media. The thermalsensitivity of Alls/Gifford (Curlicue) strain, however, was not alteredby addition of CO₂ or bicarbonate under the same conditions. Carbondioxide was added to growth conditions using techniques well known inthe art (CO2 gas generator placed in a zip-lock bag with the culturemedium plates during incubation). Dry spores of Sterne strain andAlls/Gifford (Curlicue) strain were exposed to 125° C. for variouslengths of time. FIG. 3 illustrates the thermal response of Sternestrain on blood agar (as indicated by circles) and on 4×3AT media (asindicated by squares) without CO2. FIG. 4 illustrates the thermalresponse of Sterne strain on blood agar (as indicated by circles) and on4×3AT media (as indicated by squares) with CO2. FIGS. 3 and 4 show thatthermal sensitivity of Sterne on 4×3AT media was altered by the presenceof carbon dioxide. Alls/Gifford (Curlicue) strain, however, did notdisplay this difference in thermal sensitivity.

Example 8 Bicarbonate/CO2 Control

Bicarbonate/CO2 control over growth response was further examined asfollows. Alls/Gifford strain was grown in 3AT medium with and withoutbicarbonate and the results compared with growth of Sterne strain underthe same conditions. First, a suspension of spores of each bacillusstrain was prepared in phosphate buffered saline (PBS; pH 7.4). Todetermine the initial colony forming units, each was diluted 10-fold toa 1:10⁶ dilution by transferring 50 μL of suspension into 4504 of PBS. A1-μL calibrated loop was used to streak sheep blood agar or 4×3AT agar(4× contains 2× the ingredients of 2×3AT agar). Fifty milliliters ofTSB, 2×3AT, or 2×3AT with bicarbonate (2 g/l sodium bicarbonate) wereeach placed in a 250-ml flask. Each was inoculated with 50 μL of the1:10 dilution of the respective bacillus. The flasks were incubated in ashaker incubator at 37° C. Phase contrast microscopic examination ofcolonies performed after 24 hours in liquid medium, indicated thatbicarbonate accelerates spore formation in Sterne, but not inAlls/Gifford.

Example 9 Lethality

A study was conducted comparing response of laboratory mice to infectionwith Alls/Gifford (Curlicue) strain compared to the Sterne strain. Micewere injected subcutaneously with spore (one group with Sterne sporesand another with Alls/Gifford (Curlicue) spores). Results show thatAlls/Gifford strain kills mice at the same high dose as Sterne strain(1×10⁶), but at a much delayed rate; Alls/Gifford starts killingapproximately 24 hours later than Sterne. This result is illustrated inFIG. 5. The squares represent Sterne-infected mice; diamonds,Alls/Gifford-infected mice. TTD (time to death) is shown to be laterfollowing infection with Alls/Gifford strain.

1. A composition comprising: about 55 g of trypticase soy broth (TSB)base; about 12 g potassium nitrate; about 100 mg luminol (5-amino-23-dihydro-1; 4-phthalazinedione); and about 80 mg 3-amino-L-tyrosinedihydrochloride per liter of water.
 2. A method comprising: providing asa medium the composition of claim 1; and inoculating said medium with apaternal bacteria strain; whereby a mutated bacteria strain is produced.3. The method of claim 2 wherein said paternal bacteria strain isBacillus anthracis.
 4. The method of claim 3 further comprising:introducing cells of said mutated bacteria strain into a laboratoryanimal; and producing delayed onset of death in said laboratory animal,compared to an animal into which said paternal bacteria strain isintroduced.
 5. The method of claim 2 wherein said paternal bacteriastrain is the Sterne strain of Bacillus anthracis.
 6. A methodcomprising: culturing in a medium a cell of the mutated strain of claim2, under conditions permitting expression of protein products; andpurifying one or more of said protein products from said cell or mediumof said cell.